A Green Analytical Method for Identification and Quantification of Carcinogenic Impurity N-Nitroso-Desformyl Riociguat in Riociguat Formulations by UHPLC-MS/MS

Abstract

Riociguat, a vasodilatory drug for treating pulmonary hypertension (PH), might generate a Nitrosamine drug substance-related impurity (NDSRI), reported as N-Nitroso-Desformyl Riociguat (NNDFR); a similar class of nitrosamine impurities, which are specific to each drug and are structurally related to the active pharmaceutical ingredient (API) either or both sharing a common molecular backbone but differing by specific functional groups. Such impurities have drawn growing regulatory attention due to their recognized genotoxic and carcinogenic potential, in which the identification and quantification in the product is mandatory. Thus, a sensitive and reliable Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometric (UHPLC-MS/MS) method is developed employing a Shimpack-GIS CN column (150 mm x 4.6 mm x 3 µm) with a gradient of 0.1% formic acid in water, and 0.1% formic acid in methanol for chromatography separation, and multiple reaction monitoring (MRM) mode in an electrospray ionization (ESI) environment. Optimized conditions focused on chromatographic selectivity, ionization efficiency, and matrix interference reduction. Method validation for the identification of NNDFR in the Riociguat Drug Product was confirmed that this method achieved baseline separation of NNDFR isomers with retention times of 10.037 (Isomer-1) and 10.579 (Isomer-2) minutes, prohibiting interferences from other compounds. Validation also confirmed excellent linearity (R² > 0.99), precision (RSD < 1.3%), and accuracy (recoveries from 100.5% to 110.4%). LOD and LOQ were determined to be 0.4 ppm and 1.22 ppm, respectively. A 24-hour stability study confirmed the analyte integrity, method robustness under various flow rates, column temperatures, and filtration conditions, ensuring consistent performance. This validated UHPLC-MS/MS method is robust, sensitive, and reliable for routinely quantifying NNDFR in Riociguat drug products. Subsequently, the analytical method developed was assessed for environmental sustainability using established greenness assessment tools. The AGREE score was calculated at 0.73, AGREEprep at 0.69, and the BAGI value at 70.0. These values reflected a favorable environmental profile, efficient sample preparation, and practical sustainability. Together, these metrics confirmed the method’s suitability for both regulatory compliance and routine application in industrial Quality Control laboratories. Key Words: Electrospray Ionization, Greenness, Ion Focus Interface, N-Nitroso-Desformyl Riociguat, Carcinogenic, Riociguat, Liquid–Liquid extraction.